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Chapter 12. DNA Technology The DNA Toolbox • Sequencing of the human genome was completed by 2007. • DNA sequencing has depended on advances in technology, starting with making recombinant DNA. • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule. • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes. • DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products. DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning. DNA Cloning and Its Applications • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids. • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome. • Cloned genes are useful for making copies of a particular gene and producing a protein product. • Gene cloning involves using bacteria to make multiple copies of a gene. • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell. • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA. • This results in the production of multiple copies of a single gene. DNA Cloning Bacterium 1 Gene inserted into Cell containing gene of interest plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Gene of interest 2 2 Plasmid put into bacterial cell Recombinant bacterium DNA of chromosome Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of Interest Copies of gene Basic research on gene Gene for pest resistance inserted into plants Protein harvested 4 Basic research and various applications Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes (제한효소) cut DNA molecules at specific DNA sequences called restriction sites. • A restriction enzyme usually makes many cuts, yielding restriction fragments. • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments. • DNA ligase is an enzyme that seals the bonds between restriction fragments. Restriction site DNA 5 3 1 3 5 Restriction enzyme (BamHI) cuts sugarphosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector. • A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there. Hummingbird cell TECHNIQUE Bacterial cell lacZ gene ampR gene Bacterial Restriction site Sticky ends plasmid Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome. • A genomic library that is made using bacteriophages is stored as a collection of phage clones. Foreign genome cut up with restriction enzyme or Recombinant phage DNA Bacterial clones (a) Plasmid library Recombinant plasmids Phage clones (b) Phage library • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert. • BACs are another type of vector used in DNA library construction. Large plasmid Large insert with many genes BAC clone (c) A library of bacterial artificial chromosome (BAC) clones • A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell. • A cDNA library represents only part of the genome — only the subset of genes transcribed into mRNA in the original cells. DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA Primer strand Degraded mRNA DNA polymerase cDNA Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene. • This process is called nucleic acid hybridization. • A probe can be synthesized that is complementary to the gene of interest. • For example, if the desired gene is 5 … G G C T A A C T T AG C … – Then we would synthesize this probe 3 C C G A T T G A A T C G 5 3 Radioactive DNA probe Single-stranded DNA Mix with singlestranded DNA from genomic library Base pairing indicates the gene of interest · The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest. · Once identified, the clone carrying the gene of interest can be cultured. TECHNIQUE Radioactively labeled probe molecules Multiwell plates holding library clones Probe DNA Gene of interest Single-stranded DNA from cell Film • Nylon membrane Nylon Location of membrane DNA with the complementary sequence Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA. • A three-step cycle — heating, cooling, and replication — brings about a chain reaction that produces an exponentially growing population of identical DNA molecules. TECHNIQUE Genomic DNA 1 2 Cycle 1 yields 2 molecules 5 3 3 Target sequence 5 Denaturation 5 3 3 5 Annealing Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence PCR Process 5 TECHNIQUE 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Cycle 1 yields 2 molecules Genomic DNA 3 5 5 3 3 5 1 Heat to separate DNA strands Target sequence 3 5 5 2 Cool to allow primers to form hydrogen bonds with ends of target sequences 3 5 3 DNA polymerase adds nucleotides to the 3 end of each primer 5 5 3 5 3 Primer 5 New DNA 3 Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to – Compare genes and alleles between individuals. – Locate gene expression in a body. – Determine the role of a gene in an organism. • Several techniques are used to analyze the DNA of genes. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis. • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size. • A current is applied that causes charged molecules to move through the gel. • Molecules are sorted into “bands” by their size. TECHNIQUE Mixture of DNA molecules of different sizes Power source Anode + – Cathode Gel 1 Power source – + Longer molecules 2 Shorter molecules Mixture of DNA fragments of different sizes Power source Longer (slower) molecules Gel Completed gel Shorter (faster) molecules • A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization. • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel . TECHNIQUE DNA + restriction enzyme Restriction fragments I II III Nitrocellulose membrane (blot) Heavy weight Gel Sponge I Normal II Sickle-cell -globin allele allele III Heterozygote 1 Preparation of restriction fragments Alkaline solution 2 Gel electrophoresis Paper towels 3 DNA transfer (blotting) Radioactively labeled probe for -globin gene I II III Probe base-pairs with fragments Fragment from sickle-cell -globin allele Fragment from normal -globin Nitrocellulose blot allele 4 Hybridization with radioactive probe I II III Film over blot 5 Probe detection RFLPs can be used to detect differences in DNA sequences – Single nucleotide polymorphism (SNP) is a variation at one base pair within a coding or noncoding sequence – Restriction fragment length polymorphism (RFLP) is a variation in the size of DNA fragments due to a SNP that alters a restriction site • In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis. • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene. • The procedure is also used to prepare pure samples of individual fragments. Normal -globin allele 175 bp DdeI 201 bp DdeI Normal Sickle-cell allele allele Large fragment DdeI DdeI Large fragment Sickle-cell mutant -globin allele Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene 201 bp 175 bp 376 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles Restriction enzymes added DNA sample 1 DNA sample 2 w Cut z x Cut Cut y y Longer fragments z x Shorter fragments w y y Forensic Evidence and Genetic Profiles • An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids. • Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains. • Genetic profiles can be analyzed using RFLP analysis by Southern blotting. • Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences. • STR analysis compares the lengths of STR sequences at specific regions of the genome • PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths. • The probability that two people who are not identical twins have the same STR markers is exceptionally small. • Current standard for DNA profiling is to analyze 13 different STR sites. STR site 1 STR site 2 Crime scene DNA Number of short tandem repeats match Suspect’s DNA Number of short tandem repeats do not match Crime scene DNA Suspect’s DNA (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. DNA profiling has provided evidence in many forensic investigations ● Forensics – Evidence to show guilt or innocence ● Establishing family relationships – Paternity analysis ● Identification of human remains – After tragedies such as the September 11, 2001, attack on the World Trade Center ● Species identification – Evidence for sale of products from endangered species DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain termination method. • Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths. • Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment. • The DNA sequence can be read from the resulting spectrogram. TECHNIQUE DNA (template strand) Primer DNA polymerase Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP TECHNIQUE DNA (template strand) Labeled strands Shortest Direction of movement of strands Longest Longest labeled strand Detector Laser RESULTS Shortest labeled strand Last base of longest labeled strand Last base of shortest labeled strand 그림 12.15 그림 12.15 DNA 염기서열 분석 Genomics is the scientific study of whole genomes ● Genomics is the study of an organism’s complete set of genes and their interactions – Initial studies focused on prokaryotic genomes – Many eukaryotic genomes have since been investigated ● Evolutionary relationships can be elucidated – Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans – Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast The Human Genome Project revealed that most of the human genome does not consist of genes – Goals of the Human Genome Project (HGP) – To determine the nucleotide sequence all DNA in the human genome – To identify the location and sequence of every human gene Results of the Human Genome Project Humans have ~21,000 genes in 3.2 billion nucleotide pairs ● Only ~1.5% of the DNA codes for proteins, tRNAs, or rRNAs ● The remaining 88.5% of the DNA contains – Control regions such as promoters and enhancers – Unique noncoding DNA – Repetitive DNA – Found in centromeres and telomeres – Found dispersed throughout the genome, related to transposable elements that can move or be copied from one location to another ● Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (15%) The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly Three stages of the Human Genome Project ● – A low-resolution linkage map was developed using RFLP analysis of 5,000 genetic markers – A physical map was constructed from nucleotide distances between the linkage-map markers – DNA sequences for the mapped fragments were determined Whole-genome Shotgun Method ● ● Restriction enzymes were used to produce fragments that were cloned and sequenced Computer analysis assembled the sequence by aligning overlapping regions Chromosome Chop up with restriction enzyme DNA fragments Sequence fragments Align fragments Reassemble full sequence Proteomics is the scientific study of the full set of proteins encoded by a genome ● Proteomics – Studies the proteome, the complete set of proteins specified by a genome – Investigates protein functions and interactions ● The human proteome may contain 100,000 proteins Analyzing Gene Expression • Nucleic acid probes can hybridize with mRNAs transcribed from a gene. • Probes can be used to identify where or when a gene is transcribed in an organism. Studying the Expression of Single Genes • Changes in the expression of a gene during embryonic development can be tested using – Northern blotting. – Reverse transcriptase-polymerase chain reaction. – In situ hybridization. • The methods are used to compare mRNA from different developmental stages. • Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane. • Identification of mRNA at a particular developmental stage suggests protein function at that stage • Reverse transcriptase-polymerase chain reaction (RTPCR) is quicker and more sensitive. • Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest. • The products are run on a gel and the mRNA of interest identified TECHNIQUE 1 cDNA synthesis mRNAs cDNAs 2 PCR amplification Primers -globin gene 3 Gel electrophoresis RESULTS Embryonic stages 1 2 3 4 5 6 • In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism. Studying the Expression of Interacting Groups of Genes • Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays. • DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions. TECHNIQUE 1 Isolate mRNA. Tissue sample 2 Make cDNA by reverse mRNA molecules 3 Apply the cDNA mixture to a Labeled cDNA molecules (single strands) DNA fragments representing specific genes transcription, using fluorescently labeled nucleotides. microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. DNA microarray 4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. DNA microarray with 2,400 human genes Green Fluorescent Protein • The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has become one of the most important fluorescent labels in biology. Its Applications as a Biomarker Monitoring Cellular Proteins